A major roadblock in HIV vaccine development is our inability to elicit broadly neutralizing antibodies (BNA) that recognize the HIV-1 envelope. This reflects, in part, the sequestration of key neutralizing epitopes on HIV-1 Env. One way of solving this problem is to develop antigenic mimics of these sequestered, conformational epitopes, which can then be used as immunogens to elicit BNA. Previous efforts to produce such mimics have been unsuccessful, largely because the linear mimotopes that were selected using conventional phage display technology failed to recapitulate the structure of discontinuous, virus-neutralizing epitopes on Env. We propose an innovative solution to this problem, by exploiting a unique molecular scaffold that can be diversified in three discontinuous, but interacting, surface exposed loops. Our hypothesis is that it is necessary to use a diverse, discontinuous molecular library to identify antigenic mimics of conformational gp120 epitopes such as the broadly neutralizing epitope recognized by the b12 Mab, and that these antigenic mimics can then be used to elicit broadly neutralizing antibodies against HIV-1. Experiments will be conducted to identify mimics of a discontinuous neutralizing epitope in the CD4 binding site of the HIV-1 envelope, recognized by Mab b12. To do this, we will screen diversified discontinuous protein libraries, constructed using a unique display scaffold, and presented on the surface of bacteriophage. We will then derive mimotopes that bind with high affinity to Mab b12, by using alanine-scanning mutagenesis and affinity selection. We will perform competition ELISA experiments to test whether these proteins can compete with oligomeric HIV-1 Env for binding to Mab b12. The immunogenicity of these proteins will then be assessed in rabbits, to determine if they elicit Env-specific antibody responses (including virus-neutralizing antibodies). These experiments are expected to provide a proof-of-concept evaluation of our hypothesis. The use of a novel scaffold to display multiple discontinuous peptides that can better preserve the conformation of the b12 epitope to induce b12-like BNA is a powerful, innovative approach that has not been explored previously. Moreover, if this work is successful, the same scaffold could be used for other conformational epitopes, in addition to b12. Thus, this proposal delineates a high risk but very high reward vaccine strategy - consistent with the intent of the R21 grant mechanism. PUBLIC HEALTH RELEVANCE: This application seeks to develop an improved method to produce vaccines for human immunodeficiency virus type-1 (HIV-1), by taking an innovative approach to the generation of broadly neutralizing antibodies (BNA) that recognize the HIV-1 envelope. We propose to use a novel method to produce novel antigenic mimics of key conformational epitopes on the HIV-1 envelope. We will then use these antigenic mimics to elicit broadly neutralizing antibodies capable of inhibiting the infectivity of HIV-1.